One of the defense mechanisms against infection by both animals and plants is the production of peptides that have antimicrobial and antiviral activity. Various classes of these peptides have been isolated from tissues of both plants and animals. PCT application WO 95/03325 (published Feb. 2, 1995) contains a review of the literature on this subject. Such peptides include tachyplesins, which are 17-18 amino acid peptides containing four invariant cysteines, the defensins, .beta.-defensins, and insect defensins, which are somewhat longer peptides characterized by six invariant cysteines and antifungal and antibacterial peptides and proteins which have been found in plants.
The parent applications provide a new class of antimicrobial peptides, designated "protegrins," representative members of which have been isolated from porcine leukocytes. The protegrin peptides, which are generally amphiphilic in nature, exhibit broad spectrum antimicrobial activity. Thus, these peptides are useful as antibacterial, anti-fungal and antiviral agents in both plants and animals.
The isolation of some of the protegrin peptides of the invention was reported by the present applicants in a paper by Kokryakov, V. N. et al., 1993, FEBS Lett 337:231-236 (July issue). A later publication of this group described the presence of a new protegrin whose sequence, and that of its precursor, was deduced from its isolated cDNA clone. Zhao, C. et al., 1994, FEBS Lett 346:285-288. An additional paper disclosing cationic peptides from porcine neutrophils was published by Mirgorodskaya, O. A. et al., 1993, FEBS Lett 330:339-342. Storici, P. et al., 1993, Biochem Biophys Res Comm 196:1363-1367, report the recovery of a DNA sequence which encodes a pig leukocyte antimicrobial peptide with a cathelin-like prosequence. The peptide is reported to be one of the protegrins disclosed hereinbelow. Additional publications related to protegrins are Harwig, S. S. L., et al., 1995, J Peptide Sci 3:207; Zhao, C., et al., 1995, FEBS Lett 376:130-134; Zhao, C. et al., 1995, FEBS Lett 368:197-202; Miyakawa, Y. et al., 1996, Infect Immun 64:926-932; Yasin, B. et al., 1996, Infect Immun 64:709-713; Qu, X -D et al., 1996, Infect Immun 64:1240-1245; Aumelas, A. et al., 1996, Eur J. Biochem 237:575-583; Mangoni, M. E. et al., 1996, FEBS Lett 383:93-98; Steinberg et al., 1996, "Protegrins: Fast Acting Bacterial Peptides," presented at 8th Intl. Symposium on Staphylococci and Staphylococcal Infections, Aix les Bains, France, Jun. 23-26, 1996; Steinberg et al., 1996, "Broad Spectrum Antimicrobial Activity of Protegrin Peptides," presented at 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, La., Sep. 15-18, 1996; Kung et al., 1996, "Protegrin Protects Mice From Systemic Infection By Antibiotic-Resistant Pathogens," presented at 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, La., Sep. 15-18, 1996; and Steinberg et al., 1996, "In Vitro Efficacy of Protegrins Against Helicobacter Pylori," presented at 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, La., Sep. 15-18, 1996.
The protegrins have also been found to bind to endotoxins--i.e., the lipopolysaccharide (LPS) compositions derived from Gram-negative bacteria which are believed responsible for Gram-negative sepsis. The protegrins are also effective in inhibiting the growth of organisms that are associated with sexually transmitted diseases such as Chlamydia trachomatis and Neisseria gonorrhoeae.
The present invention is directed to a new set of protegrins which offer properties of being rapid acting microbicides having a broad spectrum of activity with a low likelihood of resistance. In addition, the class of protegrins of the present invention offers an additional opportunity to adjust the spectrum of activity with respect to the type of microbe or virus most effectively inhibited and with respect to the conditions under which this inhibition occurs. The protegrins in this case differ from those of the parent applications either by deletion of at least one of the four N-terminal amino acids, or of certain other designated residues and/or by replacement of certain amino acids with those of other classes.